Expanded utility of red-cell derived microparticles (rmp) for treatment of bleeding

ABSTRACT

Red blood cell membrane derived microparticles (RMP) are safe, economical, effective hemostatic agents in the treatment of a wide range of bleeding conditions and can, therefore, be considered as universal hemostatic agents. Effective RMP are produced from red blood cells using a high-pressure extrusion membrane shear process. The RMP can be lyophilized after production and retain activity even when stored at room temperature. RMP can be administered to original donors (autologous treatment), thus avoiding transfusion complications, or can be administered to blood type compatible recipients. RMP produced from type O, Rh negative red cells can be given to any person regardless of blood type. RMP can be administered to reduce excessive bleeding resulting from trauma, surgeries, invasive procedures and various bleeding disorders such as platelet disorders, either congenital or acquired, and coagulation disorders, either congenital or acquired. Administration of RMP prepared according to the invention demonstrates effectiveness in safely reducing bleeding.

CROSS-REFERENCE TO PRIOR APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 13/357,106, filed Jan. 24, 2012 which claims benefit and priorityfrom U.S. Provisional Application No. 61/457,203, filed on Jan. 28, 2011and is also a continuation-in-part of and claims benefit and priorityfrom U.S. patent application Ser. No. 11/792,399, filed Jun. 6, 2007,now U.S. Pat. No. 8,105,632, issue date of Jan. 31 2012, whichapplication was the U.S. National Phase of PCT/US2005/044064, filed Dec.7, 2005, which was based on U.S. Provisional Patent Application No.60/633,417, filed Dec. 7, 2004; all of the aforementioned applicationsare incorporated herein by reference.

U.S. GOVERNMENT SUPPORT

N/A

BACKGROUND OF THE INVENTION

Area of the Art

The present invention is in the area of hematology and more specificallyin the area of novel treatment for bleeding.

Description of the Background

The invention relates to improved compositions comprising red cellmembrane-derived microparticles (RMP) that enhance blood coagulation,platelet activity, and promote blood clot formation and to a method fortreating excessive bleeding including but not limited to those due todisorders of platelets and blood coagulation and to methods formanufacturing such compositions. The inventive compositions are usefulin minimizing blood loss in a mammal, in particular in patientsundergoing surgical or medical invasive procedures and those with traumawhere blood loss can be substantial. RMP correct hemostaticabnormalities arising from blood clotting factor deficiencies, as wellas from deficiency in platelet numbers (thrombocytopenia) and/orfunction (platelet dysfunction).

Excessive bleeding is among the most common of life-threateningcomplications in trauma and bleeding complications in both clinics andhospitals. The bleeding patient poses a major medical challenge in allmedical specialties such as surgery, trauma, obstetrics/gynecology,cardiology, neurology, hematology, etc. At present, transfusion ofbanked blood products is the mainstay of treatment for excessivebleeding, but transfusion is very expensive [1] and carries risks ofserious short- and long-term complications.

Timing is critical in bleeding patients. Prompt intervention isessential to patient management, but often many hours are required totype, cross-match, and deliver blood from the blood bank to the patient.Therefore, blood transfusion as presently employed often fails to savethe lives of many bleeding victims. Furthermore, since blood productsmust often be given before the cause of bleeding is identified,transfusion may fail to arrest bleeding and merely replaces lost bloodwhile the bleeding continues. Days or weeks of investigation may berequired to find the underlying cause of excessive bleeding.

Treatments for bleeding differ depending on etiology of the bleeding.For example, (1) when excessive bleeding develops due to low plateletcounts (thrombocytopenia) platelet transfusion or other measures toraise platelet counts must be used to arrest bleeding. In the case ofimpaired platelet function (dysfunction) treatment to improve plateletfunction or platelet transfusion are employed. (2) In the case ofcoagulation disorders, in which one or more of 13 clotting factors arelow in level or are defective or inhibited, missing clotting factorsmust be supplied to arrest bleeding. In hemophilia A, factor VIII mustbe administered whereas in hemophilia B factor IX must be administered.Without these specific therapies to correct underlying etiology,bleeding will not stop and patients will be exposed to endlesstransfusions.

To save the lives of bleeding victims, we need agents that can beadministered safely and immediately, at reasonable cost. An idealproduct could be administered to patients on a moment's notice,regardless of the underlying etiology of the bleeding. No such agent isyet available in spite of a century-long search.

New and better products are urgently needed to promptly arrest bleedingin all situations regardless of the cause of bleeding. Such productswill save many lives and will avoid needless transfusions and associatedcomplications. Our RMP product meets all requirements of safety andefficacy required of a cost effective universal hemostatic agent. RMPcan be infused at a moment's notice and is effective in the treatment ofmost bleeding conditions. It is also expected to be less costly andsafer compared to other products intended for this purpose.

As already explained, blood can be a life-saving resource, but blood isbecoming increasingly scarce and expensive due to rising demand, limitedsupply, and more stringent regulations. According to the National BloodData Resource Center, 4.5 million people receive transfusion annually.The cost for red cell transfusions alone is $24 billion per year. Thisdoes not include platelets and other blood products. The hospital costfor transfusion-related adverse effects exceeds $10 billion per year[1]. Transfusions are associated with many short- and long-termcomplications including anaphylaxis, hemolytic reactions, transfusioninduced immune suppression, graft-versus host disease, transfusionrelated acute lung injury (TRALI) and transmission of pathogens such ashepatitis, HIV and prion diseases (mad cow disease).

This situation can only get worse with increasing age of the population.For example, to prevent heart attacks, strokes and other thromboses thataffect the elderly, increasing numbers of the patients are being treatedwith anticoagulants or antiplatelet therapy. The former includesCoumadin, Heparin, LMWH (low molecular weight Heparin), fondaparinux(Arixtra) and a new generation of oral thrombin or FXa inhibitors such aas dabigatran (Pradaxa) and rivaroxaban (Xarelto). The later includeaspirin, Plavix (clopidogrel) and other antiplatelet drugs. All thesenew anticoagulants and antiplatelet medications have serious sideeffects of promoting bleeding and, thus, increase bleeding complicationand, hence, the demand for more transfusions.

Some of the older drugs such as Coumadin and Heparin have antidotes.Therefore, bleeding from overdose of Coumadin can be treated withvitamin K, and Heparin can be neutralized by an antidote, such asprotamine, to thereby limit bleeding. However, there is no effectiveantidote for new anticoagulants such as low molecular weight heparin,e.g., Lovenox (enoxaparin) (which can be partially reversed byprotamine) and Fragmin (dalteparin), Arixtra (fondaparinux), Pradaxa(dabigatran) and Xarelto (rivaroxaban) and for most antiplatelet drugs(e.g., aspirin, Plavix and their analogs). Therefore, bleeding resultingfrom new anticoagulants and antiplatelet drugs imposes new challenges inpatient management. At present, this bleeding is treated blindly withtransfusion of blood/blood products. RMP administration can correct orimprove coagulation abnormalities induced by both the new and oldertypes of “blood thinners” as well as antiplatelet drugs as is shown inFIGS. 4A, 4B, 4C, 4D, 4E and 4H.

Although transfusion of banked blood is a mainstay of therapy forbleeding, other measures have been advocated, such as antifibrinolyticagents, DDAVP [2], but these treatments are not widely used becausetheir efficacy is unproven. Recombinant Factor Vila (i.e., NovoSeven)gained much attention and showed great promise [3], but its use islimited by prohibitive cost (e.g., in excess of $1 million for a singlepatient with high levels of FVIII inhibitors) and by reports of seriousthrombotic complications. RMP has great potential for wide applicationin in various bleeding disorders and corrects hemostatic abnormalitiesinduced by most anticoagulants including new generation anticoagulantsas well as by most antiplatelet drugs. It can be stored in ambulancesfor use in emergency medicine (e.g., at accident sites) and in operatingrooms, clinics, dental offices, pharmacies and hospitals, since it isstable at room temperature, is reasonable in cost and shows no sign ofadverse effects.

Platelet MP (PMP) and lyophilized whole platelets (LyoPLT) [4] have alsobeen proposed for treatment of bleeding. Lyophilized platelets (LyoPLT)are under current study but may be impractical compared to RMP due to(i) the high costs of scarce platelets, (ii) risk of thrombogenesis, and(iii) immuno-reactivity. The total volume of circulating platelets inblood is only 20 ml, about 1/250 that of red cells, so starting materialis costly and scarce. Platelets are highly immunogenic due to HLA, ABO,Rh, and platelet-specific antigens, which are impractical to crossmatch, hence adverse reactions are frequent. Furthermore, platelets areknown to carry tissue factor (TF) which is thrombogenic. In contrast,RMP have none of these disadvantages.

SUMMARY OF THE INVENTION

The present invention (RMP) shows significant advantages over existingtreatment options. In contrast to blood bank products, (a) RMP haveindefinite shelf-life with room temperature storage and does not need tobe stored in blood banks; (b) RMP produced from type O Rh negative redcells (universal RMP) can be administered immediately withoutcross-matching; and (c) RMP can often substitute for blood-bankproducts. In addition, use of autologous RMP (made from patient's ownblood), can be used to eliminate major risks of transfusioncomplications.

RMP have many advantages as hemostatic agents, inter alia:

(1) Ease and economy of production. Red cells (RBC) are by far the mostabundant blood cells, assuring an essentially unlimited and economicalsource for RMP production. A single conventional blood donation (500 mL)is sufficient to produce RMP to treat at least two patients. Out-datedRBC in the blood bank, which is otherwise discarded, can be used as asource of RMP production.

(2) Minimal immune reactions. RBCs are the least immunogenic and safe totransfuse to type compatible recipients. RMP produced from universaldonors (type O, Rh negative) can be stored and safely infused intopatients of any blood type. This is not the case of other blood cellssuch as platelets.

(3) Autologous option. RMP can be made from the patient's own blood andinfused back to the original donors when they bleed or are at high riskof bleeding. The use of autologous RMP will eliminate complications ofallogeneic blood transfusion. This option is well suited to patients whoanticipate bleeding problems such as prior to surgery or diagnostic ortherapeutic invasive procedures or chemotherapy which often induces bonemarrow failure and severe thrombocytopenia. Systemic diseases may alsoresult in thrombocytopenia. Those who take anticoagulants orantiplatelet drugs or agents frequently suffer bleeding complications.They can prepare their own autologous RMP to be used safely in case ofbleeding. In addition, religious groups which refused normal transfusioncould benefit by this option.

(4) Universal RMP. In emergency situations there is no time to type andcross match. RMP produced from type O and Rh negative red cells can beinfused promptly to any recipient, regardless of blood type.

For reason of safety, donors for “universal RMP” should be screenedcarefully to ensure that they are negative for hepatitis, HIV, CMV orother transmittable infections. Healthy universal donors can donateblood as often as monthly. Accordingly ample resources are available foruniversal RMP.

RMP produced by the methods described herein can be used fresh orstored. Fresh RMP can be made in local blood banks or laboratories anddistributed to operating rooms, clinics, hospitals and other medicalsettings. RMP can be stocked almost anywhere including in pharmacies,ambulances, operating rooms, clinics and hospitals. Initial work on RMPproduced the product by various types of red cell membrane disruptionsuch as sonication and treatment with ionophores. However, these methodswere cumbersome, difficult to scale up and might produce RMP that weresuboptimal. We have now developed a method of producing RMP usinghigh-pressure membrane shear technology. This method produces RMP freeof any additives and is readily scalable and easy to implement.

Using our improved methodology a supplier of RMP (such as apharmaceutical company) can produce a large quantity of universal RMP orblood type specific RMP, lyophilize them and market them as hemostaticagents.

Emergency rooms, trauma centers can treat accident victims (e.g. fromvehicular accidents). In the military, RMP can be used forfirst-responder management of battlefield wounds. RMP can be used totreat and manage sport induced trauma or injury, and for injuries innatural disasters such as earthquakes or hurricanes etc.

The use of RMP will reduce the strain on limited supplies at bloodbanks. In addition, since RMP produced from expired blood have beenfound to be as effective as RMP from very fresh blood, their productionwill not place added burden on supplies. It is anticipated that RMP willreplace the need for transfusion in many situations. Since RMP haveunlimited shelf-life, and can be produced economically, their use isexpected to be at least cost-competitive with currently used bloodproducts, resulting in an overall substantial saving in health carecosts.

DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing thrombin generation by RMP as wells as bymicroparticles from other cells (note that the pattern of thrombingeneration differs among RMP, PMP (platelet MP), EMP (endothelial MP) interms of incubation time and amplitude of thrombin generation);

FIG. 2 is a bar graph demonstrating the relative absence of TissueFactor (TF) expression on RMP as compared to microparticles derived fromother cell types;

FIG. 3A is a bar graph demonstrating hemostatic effect of RMP producedusing a high pressure extrusion device French Press (note that RMPshortened rat tail bleeding time and blood loss in rats with plateletdysfunction induced by the anti-platelet drug Plavix;

FIG. 3B is a bar graph demonstrating hemostatic effect of RMP producedusing high-pressure extrusion devices from Avestin or Constant Systems(note that RMP produced by these two devices shortened rat bleeding timein a manner similar to those produced by French Press);

FIGS. 4A-H show TEG profiles of bleeding disorders in the presence orabsence of RMP; FIG. 4A: Thrombocytopenia from aplastic anemia (plateletcount 1,000/pL); FIG. 4B: Thrombocytopenia from ITP (platelet count70,000/pL); FIG. 4C: Platelet dysfunction caused by aspirin; FIG. 4D:Platelet dysfunction caused by Plavix; FIG. 4E: Coagulopathy caused byCoumadin; FIG. 4F: Coagulopathy caused by Lovenox; FIG. 4G: Coagulopathycaused by Pradaxa; FIG. 4H: Congenital hemophilia A with a mildinhibitor;

FIG. 5 shows a TEG tracing with RMP and PMP alone and combination of RMPand PMP demonstrating shortening of R time, increasing A and enhancingMA indicative of a synergistic effect from the combination;

FIG. 6 is a graph showing that RMP increases procoagulance of factorVIII deficient plasma, corroborating the results shown in Table 2;

FIG. 7A is a graph showing that RMP enhance platelet aggregation at alow dose (0.2 pM) of ADP;

FIG. 7B is a graph showing that RMP enhance platelet aggregation at alow dose (0.3 mM) of arachidonic acid; and

FIG. 8 is a bar graph showing RMP enhanced platelet adhesion.

DETAILED DESCRIPTION OF THE INVENTION

The following description is provided to enable any person skilled inthe art to make and use the invention and sets forth the best modescontemplated by the inventors of carrying out their invention. Variousmodifications, however, will remain readily apparent to those skilled inthe art, since the general principles of the present invention have beendefined herein specifically to provide methods for the production,storage and use of RMP.

Disclosed herein are unique hemostatic benefits of high-pressure shearproduced RMP as effective and safe treatments for a wide variety ofbleeding situations. Accordingly, in addition to treatment of bleedingresulting from thrombocytopenia, platelet dysfunction and surgicalprocedures and trauma, compositions and methods are provided fortreating coagulopathy induced by anticoagulant drugs or systemic diseaseor clotting factor deficiency. These and other disorders that result inbleeding are treated by the administration of an effective amount ofhigh-pressure shear RMP to a patient in need of treatment.

Effective dosages of RMP can be determined without undue experimentationby those of skill in the art and are generally expected to be between10⁶ and 10¹² particles/kg of body weight, more usually between 10⁸ and10¹⁰ particles/kg. RMP may be administered in any suitablepharmaceutical composition according to the pharmaceutical arts,including normal saline or other physiologically acceptable buffersknown to those of skill in the art, and optionally with additionaltherapeutic compounds, excipients and carriers as may be consideredadvantageous. The pH of the suspending buffer should generally be equalto or below 7.4.

RMP may be administered by any convenient and effective means known tothose of skill in the art, particularly intravenously, or by directapplication (e.g. topically, or by local injection) to a site wherehemostasis is needed or desired. Such means will be known and/or easilydetermined without undue experimentation.

Further details about pharmaceutical compositions and administration ofRMP can be found in the parent application (U.S. patent application Ser.No. 11/792,399) of the current application.

An improved method of RMP production is provided herein; this improvedmethod employs shear induced by high pressure extrusion to enableeconomically feasible, large-scale RMP production without the use ofadditives and includes a method of long-term RMP storage. For RMPproduction by high shear principle, we used a French Press, but we alsotested RMP produced by other instruments employing the same principle,such as those from Avestin and Constant Systems in vitro. These highpressure extrusion induced shear devices all generate RMP with similarprocoagulant properties in vivo as shown in FIG. 3B. Table 1 comparesthe flow cytometric markers ° measured on RMP produced by the FrenchPress with RMP produced by either a Constant System device or by anAvestin device. These results demonstrate that RMPs generated bydifferent devices using similar high pressure extrusion are similar.

TABLE 1 Production RMP markers (Counts/uL) Device Annexin V GlycophorinA Ulex europaeus French Press 8,426 27,145 42,016 Constant Systems 7,54722,271 33,286 Avestin 4,274 10,005 18,512

Example 1 Production of RMP (Laboratory Scale)

For a single batch, 30 mL packed cells from blood bank are diluted with60 mL of isotonic saline containing 1.5 mM EDTA pH 7.4. Cells are washed3 times with the same EDTA/saline by centrifuging 15 min. each at 750×gat room temp. The final resuspension is brought to volume 60 mL, whichis then drawn into the stainless-steel pressure cell of the French Press(Thermo-Electron Inc.) and then expelled at an internal pressure of25,000 psi at rate 1.5 mL/min (achieved by adjustment of the needlevalve). The resulting effluent was centrifuged at low speed (750×g) toremove the small number of unbroken cells, and the supernatant was thencentrifuged at 18,000×g for 45 min. to sediment the RMP. One of ordinaryskill in the art will appreciate that filtration and other separationtechniques can be employed to remove the unbroken cells. The RMP werethen washed at the same speed in isotonic saline (no EDTA) andrefrigerated overnight prior to lyophilization. This procedure is >99%efficient in the sense that the starting red cells are almost entirelyconverted to uniform RMP of suitable size range.

Example 2 Potential for Scale-Up

This principle (shear induced by high-pressure extrusion) can be readilyadapted to commercial-scale production. It was arrived at only afterextensive experimentation with many other methods such as ionophore,sonic disruption, osmotic rupture, and others. The basic function of aFrench Press is to apply shear to fluid suspended cells. The cellsuspension is placed in a pressure cell where pressure is applied bymeans of a hydraulic ram whereupon the pressurized suspension is forced(extruded) through the orifice of a needle valve into a region ofatmospheric pressure. The great drop in pressure as the cells passthrough the orifice applies great shear force to the biomembranes. Theprocess is controlled by setting a target pressure on the hydraulic ramand controlling the needle valve to achieve a given flow rate. Adrawback to the French Press is that the volume processed is limited bythe size of the pressure cell (typically 100 mL) and the need tomanipulate the needle valve accurately. Extensive study of FrenchPress-treated membranes has demonstrated that the system generallyproduces inside out membrane vesicle. A number of related high-pressureshear systems obviate some of the disadvantages of the French Press.Pumped fluid homogenizers pressurize a larger volume of fluid and forceit through a spring loaded valve into a region of atmospheric pressure.The effect is the same as the French Press except that the valve springcan be more reproducibly preset. Other pumped fluid homogenizersdispense with any type of valve and simply extrude the cell suspension(at a selected pressure) through a narrow channel or a fixed orificeinto a region of atmospheric pressure. Both the pressure setting and theorifice diameter or channel diameter/length can be selected to achievethe desired results. Pumping in such systems can be achieved byreciprocating piston systems so that the device is essentially aself-filling French Press. Alternatively, the effluent stream from thehigh pressure extrusion system can be directed to impinge on a surface(e.g., a metal plate) to enhance the disruption of RBC. One of ordinaryskill in the art can readily adjust the target pressure and thevalve/channel openings of such devices to achieve production of highquality RMP.

Example 3 Lyophilization for Storage

An RMP batch (from Example 1) made the previous day was mixed withoptimal amounts of albumin, glucose, sorbitol and/or trehalose, thenpipetted in 1.0 mL aliquots into 2 mL lyophilizing vials, then placed inthe tray of a Triad Freeze Dry System (Labconco, Inc.). The instrumentoperation is divided into programmable segments. Our experimentsestablished the following optimal program settings. Samples are firstsubjected to a 4-hour “Pre-freeze” phase, with brief vacuum, duringwhich temperature drops to −75° C. Segment 1. After switching to fullvacuum (<0.07 mbars), temperature is ramped up (at 0.27° C./min) to −30°C. and held for 2-hr. Segment 2. Temperature is slowly increased (at0.04° C./min) to −25° C., and held for 2-hr. Segment 3. Temperature isramped to −15° C. at 0.04° C./min, and held for 12-hr. Final Step. Vialsare sealed in vacuum; rubber stoppers were pre-placed on the vials andpressed on to seal by pressure plate. After removing vials, aluminum tabseals are crimped on; vials are stored at room temp. Activity recoveredis equal or superior to freezing at −70° C. for at least 90 days, withno decline observed. Of course, the material can be stored at lowtemperature to ensure long-term activity, but our tests indicate thateven a protracted period of storage at room temperature does not destroyactivity.

The inventors found that RMP lyophilized in this manner show excellentefficacy in vitro and in animal models, as compared with fresh RMP(prior to freezing or lyophilizing). Persons of ordinary skill in theart will appreciate that some variation in the procedural parameterswill produce an equally superior product and that further improvement inproperties of the RMP product may be made by routine adjustments of theprocedural parameters. As mentioned above, the lyophilized RMP show nodecline of activity for at least 90 days at room temperature.

Example 4 Thrombin Generation Assay (TGA) (FIG. 1)

This assay was based on the method and software of Hemker et al. [6],termed “Calibrated Automated Thrombin” (CAT) generation assay [7],adapted by the inventors to measure activity of microparticles [8]. Themethod measures a change in signal when a fluorescent substrate forthrombin is cleaved. Procedure: In the experiment shown, RMP weregenerated by exposure of washed fresh RBC from normal donor to calciumionophore A23187 (10 μM) for 1 hr. In a later study (not shown), wegenerated RMP by means of the high pressure shear extrusion methoddescribed above and confirmed that RMP produced using both methods showsimilar hemostatic activity. PMP (platelet microparticles) weregenerated by exposure of human platelet rich plasma to ADP (10 μM) for30 min. EMP (endothelial microparticles) were obtained from thesupernatant of cultured human renal microvascular endothelial cellsactivated by tumor necrosis factor (TNF-α) (10 nM) for 24 hrs. In allcases unbroken cells were removed by centrifugation (700×g for 15 min),and the resulting microparticles were sedimented (15,000×g for 30 min)and pellets were washed twice, then resuspended to equal microparticleconcentrations (1×10⁸ counts/mL, final concentration) based on counts byflow cytometry. Then 10 μL of microparticles were mixed with 90 μL ofcorn trypsin inhibitor (25 μg/mL)-treated particle free plasma (PFP)after which the CAT test was initiated by addition of calcium. Relevantdata are chiefly the lag time, seen on the x-axis, and peak thrombingenerated, seen on the y-axis in units/nM. The inventors' laboratory wasfirst to document thrombin generation by RMP [5]. The time-course ofthrombin generation by RMP is distinctive, as shown. Note the longlag-time of RMP compared to PMP and EMP, which is attributed to theabsence of tissue factor (TF). This is a great advantage of RMP ashemostatic agent. RMP, however, generate equally strong amplitude oncecoagulation is initiated. As seen in FIG. 1, the mode of thrombingeneration differed among EMP, PMP and RMP. Accordingly, combined use ofEMP and PMP along with RMP could be of benefit in certain clinicalsettings. MP types with distinct hemostatic property can be combined toexert optimal hemostatic efficacy in reducing bleeding. This is animportant rational for supporting MP combination therapy.

Example 5 Absence of TF Expression on RMP (FIG. 2)

The method used was flow cytometric detection of TF antigen usingPE/Cy5-labeled anti-TF (American Diagnostica). PMP, EMP and RMP wereprepared as described in the previous section. LMP (leukocytemicroparticles) were prepared by incubating neutrophils (5×10⁶/mL) withlipopolysaccharide (LPS, 10 ng/mL final concentration). The neutrophilswere isolated by standard procedures (Ficol-Hypaque densitycentrifugation). All microparticles were adjusted to equal concentration(1×10⁸/mL). TF was measured in flow cytometry by double-staining(anti-TF and FITC-Ulex lectin) prior to aspiration into the flowcytometer. Event detection was triggered by the green fluorescentsignal. Results are given as percentage of microparticle-positive eventsthat were also positive for TF. It is apparent that TF expression onmicroparticles would promote systemic thrombosis. However, TF canbenefit local hemostasis at the site of injury. Accordingly propercombination of RMP and tissue factor expressing microparticles will havetherapeutic advantage of certain bleeding condition as described above.

The figure demonstrates that TF is not detected on RMP but is easilydetected on other cell-derived microparticles: those from platelet(PMP), endothelia (EMP) and leukocytes (LMP). This result is consistentwith literature identifying, TF on all microparticles except t RMP. TFis the main physiological initiator of coagulation and thrombosis. Sothat the absence of TF on RMP indicates a high degree of safety, i.e.,inability to generate thrombin, except a local site of injury where TFis present. In other words, RMP are not efficient in initiating thrombingeneration, but contribute robustly to its generation upon exposure ofTF, such as at a bleeding site, thus providing effective hemostasislocally but not systemically.

Because RMP, PMP, EMP, LMP have different hemostatic property asdemonstrated in FIGS. 1 and 2, use of RMP combined with other MP such asPMP can exhibit a synergistic effect. FIG. 5 demonstrates thatcombination of RMP with PMP exhibit synergistic effect in hemostasiswhen examined by TEG. Use of the combination shortened clotting time (R)and increased speed of clot formation (A) and enhanced clot strength(MA) compared to RMP or PMP alone.

Example 6 Use of RMP to Treat Platelet Dysfunction (FIG. 3)

In the parent of the instant application, applicants presented datashowing that infusion of RMP shortened tail bleeding time inthrombocytopenic rats.

The question addressed by this study was whether RMP were able to reducebleeding from platelet dysfunction as well. We induced plateletdysfunction in rats by intravenous injection of Plavix (10 mg/kg). FIG.3 shows that platelet dysfunction induced by Plavix increased bleedingtime and blood loss by at least 5-fold compared to controls, and someanimals did not stop bleeding at all and expired. However, all eightanimals treated with RMP (dose of 2.0×10⁹/kg) showed almost completenormalization of bleeding time and blood loss.

The experiments were conducted in Sprague-Dawley rats. The procedure wasbegun by weighing the animal (range 250-350 g) and anesthetizing them byadding 1-2 mL of Isoflurane to a sealed container in which the rat wasplaced. Once sedated, the rat was placed on a special platform thatallows a technician to intubate it. The animal constantly receivedoxygen and Isoflurane from a respirator to maintain anesthesia. Theanimal was then affixed to a surgical board and a rectal thermometer isused to monitor body temperature. A heating pad was placed under theboard to maintain body temperature. When stable, the neck of the animalwas shaved and a small incision was made to find the jugular vein andthe carotid artery. Any slight bleeding during this procedure wasstopped with a cautery tool. Both the jugular vein and the carotidartery were cleaned and isolated from surrounding connective tissue.Before inserting the cannulas, blood flow was controlled by tying asection of the vein and artery. Once the cannulas were in place, theywere secured with sutures and the incision closed. The jugular vein waslater used to inject test substances (RMP, medications) and the carotidartery was used to supply vital fluids (lactated Ringer's) and tomonitor heart rate and blood pressure. This allows the rat to survivefor the required 3-4 hours. Bleeding was initiated by cutting a 2 mmsegment of a toe and all blood was collected in tube for “total bloodloss” and time to cease bleeding was measured.

Baseline data was obtained for n=23 untreated animals (controls; atleft). Platelet dysfunction was induced by infusion of clopidogrel(Plavix) at a dose of 10 mg/kg through the catheter 10 min. prior tobleeding. The center bar shows increased bleeding time and blood lossdue to clopidogrel. The error bar is +/−SEM. The left panel is acomparison of bleeding time between controls, animals treated withPlavix, and animals treated with both Plavix and RMP. The right panel isa comparison of total blood loss between the same groups. RMPadministered at a dose of 2.0×10⁸/kg in volume 400 μL five minutes priorto bleeding gave correction of bleeding time and blood loss tonear-normal range, as shown (right bars). Note the near normalization ofbleeding time and blood loss by RMP infusion.

In our animal model, improved instrumentation was used to measure heartrate, blood pressure, respiration and temperature. Vital signs wereclosely monitored before and after RMP infusion. Bleeding time and totalblood loss were measured after cutting toe, as described. All treatedanimals were observed for up to four hours post-treatment (underanesthetic). None of the RMP-treated animals showed indication ofthrombosis or signs of adverse change in temperature, heart rate,respiration, or neurological signs. This included two cases tested atdouble the full effective dose; no further reduction in bleeding time(below normal) was observed. This indicates absence of prothromboticeffects, and suggests complete safety in all cases studied thus far.Since RMP do not carry tissue factor (TF), there is no theoreticalreason to expect adverse effects. The apparent absence of adverseeffects even at high dose is considered to be an important advantage ofRMP compared to other products intended for this purpose.

We also studied the effectiveness of RMP generated by different highpressure instruments (e.g., Avestin, Constant system) using the ratmodel described above. These RMP were as effective in shortening tailbleeding time as were RMP produced by the French Press high pressureinstrument (as shown in FIG. 3A). Table 1 compares the flow cytometricmarkers measured on RMP produced by the French Press with RMP producedby either a Constant Systems device or by an Avestin device. Theseresults demonstrate that RMP produced using various high pressureextrusion devices are similar.

Example 7 Clinical Study of RMP on Various Bleeding Disorders by Meansof TEG

Bleeding time (BT) was formerly used to predict risk of bleeding atsurgery but is now replaced by thromboelastograph (TEG) measurementsbecause of superior reproducibility and the additional important dataprovided by TEG instruments [9-11]. TEG is the best in vitro assay topredict risk of bleeding in vivo and its value in assessing hemostasisis well accepted. TEG measurement is increasingly utilized to assessbleeding with invasive procedures or surgery such as renal biopsy [8],cardiovascular surgery [9, 10] and trauma [11]. It is such a widelyaccepted practice now for surgeons to pre-order blood for transfusionbased on TEG abnormality in an effort to prevent excessive bleeding.

TEG Protocol:

Blood samples were drawn in citrated vacutainers from patients withvarious bleeding disorders. Blood samples were pipetted (330 μL) intothe cup of the “TEG Haemoscope” (Haemonetics Co.), then 10 μL of saline(control, without RMP). RMP produced through high-pressure extrusion wasadded to each well at the dose of 6.8×10⁷ particles to bring the finalRMP concentration to 2×10⁸/mL. The reaction was initiated by adding 10μL of CaCl₂. TEG tracings were obtained without and with RMP, andalteration of TEG parameters by addition of RMP was evaluated. Thequantity of RMP added was calculated from the therapeutic dosedetermined in animal experiments.

FIGS. 4 A-G are examples of clinical studies to evaluate hemostaticproperty of RMP. It should be noted that TEG tracings without RMP areshown in unbolded solid lines and tracing after addition of RMP areshown in bolded broken lines. In each figure, changes in R, A and MA arecompared in the boxes next to figures. These findings indicate potentialvalue of RMP to treat various bleeding disorders. The applicants foundthat TEG gave results which corresponded closely to in vivo (animal)studies, in particular, results with Plavix, building confidence thatTEG is a good surrogate for in vivo studies. This confirms literaturethat TEG correlates closely with bleeding time in vivo (human). TEG isalso more reproducible, faster, and supplies additional importantmeasurement parameters [9-11].

Parameters Measured.

TEG measures the delay of initiation of clotting (R), the rate of clotformation (a, angular rise rate), and maximum amplitude (MA). A highrisk of bleeding is detected as a high R with a low a, or low MA measurevalue.

In aplastic anemia (FIG. 4A), bone marrow fails to produce sufficientred cells, white cells and platelets; this patient had fewer than 1% ofnormal platelets (1,000/μL vs. normal value of 250,000/μL). The figureshows almost no clotting in absence of RMP, but with a therapeutic doseof RMP a nearly normal trace is seen as indicated by fast initiation(R=9.7 min, from 53.5), a rate of rise (a=52.8, from 1.3) and anincrease of MA (from 2.2 to 45.5) as detailed in the figures' sideboxes.

In ITP (Idiopathic Thrombocytopenic Purpura) (FIG. 4B), platelets aredestroyed by an autoimmune reaction; this patient had only 28% of thenormal number of platelets. In absence of RMP, clotting is delayednearly three-fold longer than normal (R=23.9) and rate is weak as seenin the low slope (a=19.5). A normal profile is restored by therapeuticdose of RMP (R=8.3, a=61.8).

In this patient (FIG. 4C), platelet dysfunction was due to aspirin,which inhibits platelet function and can result in serious bleeding,especially in combination with other disorders or medications. Note theelimination of the prolonged lag time (R) by RMP.

In this case (FIG. 4D) platelet dysfunction was due to therapy withPlavix (clopidogrel), which inhibits platelet function as does aspirinbut by a different mechanism (blockade of ADP receptors). Despite adifferent mechanism of platelet dysfunction, RMP corrected the prolongedlag time.

Coumadin (a.k.a. Warfarin) is most widely used “blood thinners”prescribed for prevention and treatment of thrombosis. It acts bypreventing effective production of the several clotting factors thatrequire vitamin K. Overdose of Coumadin, which happens commonly, canlead to serious bleeding. In this patient (FIG. 4E), notice that RMPfully corrected the prolonged lag time (R) and slow rate (a). Plateletcounts were normal in this patient.

Many new anticoagulants will soon come to market. The old as well as thenew anticoagulants belong to one of four groups: 1) Vitamin Kantagonists such as Coumadin; 2) Heparin and low molecular weightheparin; 3) inhibitors of thrombin (anti-factorlla) such as dabigatran;and 4) inhibitors of prothrombinase complex (anti-factor Xa) such asfondaparinux and rivaroxaban. The new anticoagulants are increasinglyemployed. Low molecular Heparin (LMWH) and especially enoxaparin(Lovenox) and dalteparin (Fragmin) are now widely used. These newanticoagulants do not have antidotes, therefore hemorrhagiccomplications from new anticoagulants is very problematic. FIG. 4F showsthat RMP correct coagulation abnormality induced by Lovenox. Similarcorrection by RMP was seen in other LMWH such as dalteparin (Fragmin)and fondaparinux (Arixtra) as well as dabigatran (Pradaxa), a new oralthrombin inhibitor (FIG. 4 G) which is more convenient and practical touse than existing anticoagulants. RMP corrects clotting abnormalitiesinduced by anticoagulants from any of the four groups. Therefore, RMPshould be effective against bleeding resulting from any new drugsbelonging to one of these groups.

Hemophilia A is an inherited disorder marked by ready bleeding due tolow levels of functional factor VIII (FVIII). Painful and debilitatingbleeding in the joints and mucous membranes are common, and brainhemorrhage can be fatal. Treatment is infusion of FVIII concentrates butthis treatment is often ineffective due to formation of inhibitors ofthe FVIII administered. This patient (FIG. 4H) had congenital HemophiliaA and developed mild inhibitor. The figure shows that RMP correctedprolonged lag (R) and normalized rate (a) of clotting.

Examples 8 Mode of Action of RMP in Hemostasis

To investigate how RMP exert diverse actions in hemostasis, we mixed RMPwith factor deficient plasma and studied alteration of procoagulancewith TEG. Addition of RMP to factor deficient plasma shortened clottingtime (R), increased rated of clotting (a) and enhanced clot strength(MA). However degree of increasing procoagulance by RMP differed betweenclotting factor deficiency. Effects of RMP in Factor II, V, VII, VIII,IX, X, XI XII, XIII deficiency are shown in the Table 2 [12]. Theincrease in procoagulance by RMP was most pronounced in Factor VIII (seeFIG. 6) and Factor IX deficient plasmas where RMP could correctdeficiencies as low as 5-10% of the normal levels of the clottingfactors. In all cases addition of RMP resulted in a significantcorrection of clotting time.

Other Bleeding Conditions Seen Corrected.

RMP was observed to correct the abnormality in the following casesjudged by TEG: (i) Patients with mild inhibitors to Factor IX, XI; (ii)patients treated with heparin, low molecular weight heparin (Lovenox,Fragmin) and Arixtra; (iii) patients with DIC; (iv) chronic liverdisease; (v) thrombocytopenia from bone marrow failure such as aplasticanemia, myelodysplastic syndrome, leukemias. (data not shown).

FIGS. 7A and 7B show the effect of RMP on platelet aggregation andadhesion. At low concentrations of ADP (FIG. 7A) and arachidonic acid(FIG. 7B), RMP enhanced platelet aggregation measured by Chrono-Logaggregometry. We also studied the effect of RMP on platelet adhesion bya cone-well device (Diamed IMPACT-R), where RMP enhanced plateletadhesion by increasing the sizes of aggregates. FIG. 8 shows theenhancement in platelet adhesion. Note the statistically significant (*P=0.02) increase in size of aggregates of platelets by RMP at shear rate(S⁻¹)=1800, that resemble venous blood flow in vivo [12].

TABLE 2 RMP enhance procoagulance in factor deficient plasmas. R(min) A(degree) MA (mm) F II RMP− 8.7 63.6 33 RMP+ 8.4 68.1 37 Δ −0.3 4.5 4 F VRMP− 11.2 62.1 29.2 RMP+ 11.1 64.8 28.7 Δ −0.1 2.7 −0.5 F VII RMP− 11.657.7 29.6 RMP+ 11.3 62 25.5 Δ −0.3 4.3 −4.1 F VIII RMP− 30.8 23.1 26.3RMP+ 16.6 52.6 29.7 Δ −14.2 29.5 3.4 F IX RMP− 77.2 1.1 7.2 RMP+ 28.212.6 27.8 Δ −49 11.5 20.6 F X RMP− 11.2 62.1 29.1 RMP+ 11.1 64.8 28.7 Δ−0.1 2.7 −0.4 F XI RMP− 14.8 55.6 27.6 RMP+ 12.2 50.2 47.7 Δ −2.6 −5.420.1 F XII RMP− 13.1 32.9 25.4 RMP+ 11.1 55.3 35.2 Δ −2 22.4 9.8 F XIIIRMP− 8.5 56.2 20.8 RMP+ 8.2 57.7 24.1 Δ −0.3 1.5 3.3

The following claims are thus to be understood to include what isspecifically illustrated and described above, what is conceptuallyequivalent, what can be obviously substituted and also what essentiallyincorporates the essential idea of the invention. Those skilled in theart will appreciate that various adaptations and modifications of thejust-described preferred embodiment can be configured without departingfrom the scope of the invention. The illustrated embodiment has been setforth only for the purposes of example and that should not be taken aslimiting the invention. Therefore, it is to be understood that, withinthe scope of the appended claims, the invention may be practiced otherthan as specifically described herein.

REFERENCES

References and publications cited herein are hereby incorporated byreference

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What is claimed is:
 1. A process for producing RMP comprising the stepsof: suspending red blood cells in an aqueous diluent to form a cellsuspension; pressurizing the cell suspension; extruding the pressurizedcell suspension into a region of lower pressure to generate shear forceson the suspended red blood cells whereby the suspended cells areconverted into a crude RMP suspension; and removing any whole red bloodcells from the crude RMP suspension to make a final RMP suspension. 2.The process according to claim 1, wherein a French Press is used topressurize and extrude the cell suspension.
 3. The process according toclaim 1, wherein whole red blood cells are removed from the RMPsuspension by centrifugation.
 4. The process according to claim 3,wherein RMP are removed from the crude RMP suspension by centrifugation.5. The process according to claim 1, wherein whole red blood cells areremoved from the RMP suspension by filtration.
 6. The process accordingto claim 1 further comprising a step of washing the red blood cells withsaline prior to the step of suspending.
 7. The process according toclaim 6, wherein the saline further comprises EDTA.
 8. The processaccording to claim 1 further comprising a step of lyophilizing the finalRMP suspension.
 9. The process according to claim 1 wherein the redblood cells are from an autologous donor.
 10. The process according toclaim 1, wherein the red blood cells are selected from the groupconsisting of type A—Rh positive, type A—Rh negative, type B—Rhpositive, type B—Rh negative, type AB—Rh positive, type AB—Rh negative,type O—Rh positive and type O, Rh negative.
 11. The process according toclaim 10, wherein the red blood cells are type O—Rh negative.
 12. Aprocess for treating and preventing excessive bleeding in a mammalcaused by traumas, surgeries, invasive procedures or due to bleedingconditions including platelet or coagulation disorders, eithercongenital or acquired comprising the step of administering RMP to themammal.
 13. The process according to claim 12, wherein the plateletdisorder is either thrombocytopenia or platelet dysfunction.
 14. Theprocess according to claim 13, wherein thrombocytopenia is caused byimmune system, by drugs or agents, by chemotherapy, by systemic disease,or by bone marrow failure.
 15. The process according to claim 13,wherein the platelet dysfunction is either congenital or acquired. 16.The process according to claim 15, wherein the platelet dysfunction iscaused by a drug treatment.
 17. The process according to claim 16,wherein the drug treatment is treatment with a drug that impairsplatelet function.
 18. The process according to claim 17, wherein thedrug is selected from the group consisting of aspirin, clopidogrel,other antiplatelet drugs and other antiplatelet agents.
 19. The processaccording to claim 12, wherein the coagulation disorder is congenital oracquired.
 20. The process according to claim 19, wherein the coagulationdisorder is caused an anticoagulant selected from the group consistingof Coumadin, heparin, inhibitors of prothrombinase complex andinhibitors of thrombin including dabigartran.
 21. The process accordingto claim 20, wherein the low molecular weight heparin is enoxaparin ordalteparin.
 22. The process according to claim 20, wherein the inhibitorof prothrombinase complex is fondaparinux or rivaroxaban.
 23. A processfor treating and preventing excessive bleeding in a mammal comprisingthe step of administering RMP in combination with other cell derivedmicroparticles to the mammal.
 24. The process according to claim 20,wherein the other cell derived microparticles are PMP.